Fraxinus mandshurica Rupr. (F. mandshurica) is a dioecious tree species with important ecological and application values. To delve deeper into the regulatory pathways and genes responsible for male and female flowers in F. mandshurica, we conducted transcriptome sequencing on male and female flowers at four distinct stages. The analysis revealed that the female database generated 38,319,967 reads while the male database generated 43,320,907 reads, resulting in 2930 differentially expressed genes with 1441 were up-regulated and 1489 down-regulated in males compared to females. Following an analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), four distinct pathways (hormone signal transduction, energy metabolism, flavonoid biosynthesis, and photoperiod) linked to female and male flowers were identified. Subsequently, qRT-PCR verification revealed that FmAUX/IAA, FmEIN3, and FmA-ARR genes in hormone signal transduction pathway are related to female flower development. Meanwhile, FmABF genes in hormone signal transduction pathway, FmGS and FmGDH genes in energy metabolism pathway, FmFLS genes in flavonoid biosynthesis pathway, and FmCaM, FmCRY, and FmPKA genes in photoperiod pathway are related to male flower development. This study was the first to analyze the transcriptome of male and female flowers of F. mandshurica, providing a reference for the developmental pathways and gene expression levels of male and female plants.